Copyright Ó 2006 by the Genetics Society of America
DOI: 10.1534/genetics.106.059246
Variable Populations Within Variable Populations: Quantifying
Mitochondrial Heteroplasmy in Natural Populations of the
Gynodioecious Plant Silene vulgaris
Mark E. Welch, Michael Z. Darnell1 and David E. McCauley2
Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235
Manuscript received April 10, 2006
Accepted for publication July 31, 2006
ABSTRACT
Populations of mitochondria reside within individuals. Among angiosperms, these populations are
rarely considered as genetically variable entities and typically are not found to be heteroplasmic in nature,
leading to the widespread assumption that plant mitochondrial populations are homoplasmic. However,
empirical studies of mitochondrial variation in angiosperms are relatively uncommon due to a paucity of
sequence variation. Recent greenhouse studies of Silene vulgaris suggested that heteroplasmy might occur
in this species at a level that it is biologically relevant. Here, we use established qualitative methods and a
novel quantitative PCR method to study the intraindividual population genetics of mitochondria across
two generations in natural populations of S. vulgaris. We show incidences of heteroplasmy for mitochondrial atpA and patterns of inheritance that are suggestive of more widespread heteroplasmy at both
atpA and cox1. Further, our results demonstrate that quantitative levels of mitochondrial variation within
individuals are high, constituting 26% of the total in one population. These findings are most consistent
with a biparental model of mitochondrial inheritance. However, selection within individuals may be instrumental in the maintenance of variation because S. vulgaris is gynodioecious. Male sterility is, in part,
regulated by the mitochondrial genome, and strong selection pressures appear to influence the frequency
of females in these populations.
T
HE level of heterozygosity frequently considered in
population genetic studies represents a measure
of the within-individual component of genetic variation for biparentally inherited nuclear genes. In diploids
within-individual nuclear variation is generally limited
to two possible states, homozygote or heterozygote.
In contrast, the intraindividual component of genetic
variance with regard to the mitochondrial genome has
the potential to be far more complex because entire
populations of mitochondria reside within individuals
and are further subdivided among cells (Mackenzie
and McIntosh 1999; Birky 2001; Rand 2001). Because
of this, the magnitude of within-individual variation could
potentially be considered a continuous trait. However,
in flowering plants within-individual populations of mitochondria are widely assumed to be genetically invariant
(homoplasmic), largely as a consequence of strict maternal inheritance.
With uniparental inheritance, mutation represents
the main source of novel genetic variation within
Sequence data from this article have been deposited with the EMBL/
GenBank Data Libraries under accession nos. DQ422872–DQ422877.
1
Present address: Graduate Program in Ecology, Duke University,
Durham, NC 27708.
2
Corresponding author: Department of Biological Sciences, Vanderbilt
University, Box 1634, Station B, Nashville, TN 37235.
E-mail: david.e.mccauley@vanderbilt.edu
Genetics 174: 829–837 (October 2006)
evolutionarily independent lineages because there is
no opportunity for admixture. The thinking is that homoplasmy is enforced by the successive cell divisions
that represent sequential founder events or bottlenecks
for the mitochondrial population (Birky 2001; Rand
2001). As a result, co-occurrence of genetically distinct
mitochondrial genomes within an individual (heteroplasmy) should be rare. Further, these rare and brief
periods of heteroplasmy should be difficult to detect
with sequence data, as new mutants should be nearly
identical to the primary matrilineal haplotype (Avise
et al. 1985; Takahata and Palumbi 1985). Thus, documentation of mitochondrial heteroplasmy in natural
plant populations would suggest that either inheritance
is not strictly maternal, with the heteroplasmic state
maintained by at least occasional biparental inheritance,
or that heteroplasmy is actively maintained by some
within-individual process, such as selection favoring heteroplasmic cells or different mitochondrial haplotypes
in different tissues.
The question of whether homoplasmy and strict maternal inheritance of the mitochondrial genome are
the norm in flowering plants is relevant to several issues
in plant evolutionary biology, including the utility of
mitochondrial markers for studying gene flow by seeds
(Petit et al. 2005), the impact of mitochondrial maternal inheritance and heteroplasmy on cytonuclear
830
M. E. Welch, M. Z. Darnell and D. E. McCauley
coevolution and conflict (Wade and McCauley 2005),
the utility of cytoplasmic genes for phylogenetic studies
(Barkman et al. 2000; Wolfe and Randle 2004), maintenance and measurement of genetic diversity (Birky
et al. 1983, 1989; Rand and Harrison 1989), and issues
related to the role of recombination in the evolution of
the plant mitochondrial genome (Städler and Delph
2002). Should heteroplasmy occur in natural plant
populations at levels greater than currently thought, it
would be important to document its origins, maintenance, and mode of transmission.
While there are a small number of examples in angiosperms of biparental inheritance of the mitochondrial
genome and/or mitochondrial heteroplasmy derived
from experimental crosses (Reboud and Zeyl 1994;
Mogensen 1996; Korpelainen 2004), direct evidence
for heteroplasmy in natural populations of angiosperms
is virtually nonexistent (Barr et al. 2005). Lack of such
documentation may be due, in part, to the lack of polymorphic genetic markers that results from low levels of
sequence variation observed in the mitochondrial genomes of many plant species (Wolfe et al. 1987; Palmer
et al. 2000).
Two gynodioecious species in the genus Silene
(Caryophyllaceae) provide notable exceptions to the
general lack of mitochondrial sequence variation observed in most taxa (Städler and Delph 2002;
McCauley et al. 2005). Gynodioecy is a mating system
in which hermaphrodites and females co-occur, and
gender is typically regulated by a cytonuclear interaction involving cytoplasmic male sterility (CMS) factors
associated with the mitochondrial genome (Schnable
and Wise 1998; Dudle et al. 2001). The relative number
of females within gynodioecious populations is thought
to be under frequency-dependent selection (Frank
1989; McCauley and Brock 1998), and it is generally believed that this selective force is responsible
for maintaining mitochondrial variation that would
otherwise be lost in these gynodioecious populations
(McCauley and Taylor 1997; Laporte et al. 2001;
Bailey et al. 2003; McCauley and Olson 2003; Byers
et al. 2005).
Several recent studies have indicated that mitochondrial heteroplasmy could be relatively common in this
genus. Observations consistent with intragenic recombination of the mitochondrial genome in Silene acaulis
were considered by Städler and Delph (2002) to be
indirect evidence for heteroplasmy in that species.
Andersson (1999) has provided evidence that offspring
sex ratios in S. vulgaris differ significantly even when
generated by crosses involving a common pollen donor
and pollen recipients that consisted of female and
hermaphrodite flowers co-occurring on the same plant.
One explanation for this finding is that different parts
of the same maternal plant carry significantly different
mitochondrial populations that include different forms
of CMS.
Experimental crosses of S. vulgaris conducted by
McCauley et al. (2005) utilized PCR/RFLPs of the mitochondrial genes atpA and cox1 to document rare cases
in which offspring carried different alleles for these
genes from their respective mothers. In some instances
the offspring had the same allele as that of its father,
suggesting paternal inheritance, although in others
it did not match either parent. An explanation for the
latter observation was that the mother was heteroplasmic, carrying a secondary mitochondrial haplotype
at a level too low to visualize PCR/RFLPs on agarose gels.
It was hypothesized that occasionally a rare secondary
mitochondrial haplotype inherited by one of her offspring could become the dominant haplotype as a
result of drift and/or selection during the series of cell
divisions leading from the zygote to maturity, causing
that individual to be scored for an allele that differed
from its mother.
More direct evidence for heteroplasmy came from
instances in which PCR/RFLP banding patterns appeared to be overlays of two distinct alleles. In other
cases, readily distinguishable allelic designations of an
individual could be changed by a ‘‘knockback’’ method,
where whole genomic DNA is digested with a restriction enzyme that targets allele-specific sequence prior to
PCR/RFLP genotyping. These latter observations were
said to be cases of ‘‘cryptic heteroplasmy’’ because the
level of the putative secondary haplotype within that
individual was too low to be detected visually by the
standard PCR/RFLP method (McCauley et al. 2005).
Despite strong evidence for mitochondrial heteroplasmy from these artificial crosses, the frequency of
heteroplasmy in natural populations of S. vulgaris is
unknown, the level of quantitative genetic variance of
mitochondrial populations within individuals is unmeasured, and transmission rates of heteroplasmy from
mother to offspring have never been described. In this
study, we utilize mother–offspring arrays collected from
three natural populations of S. vulgaris to document
inheritance patterns of atpA and cox1 PCR/RFLP markers that would suggest heteroplasmy and/or biparental
inheritance. We then quantify the magnitude of heteroplasmy in terms of relative frequencies of atpA alleles within individuals, using quantitative PCR (Q-PCR),
which allows us to conduct within-individual population
genetic analyses of allele frequencies and their impact
on within-population structure, as well as their transmission rates across generations. Our findings of widespread heteroplasmy suggest that paternal leakage of
mitochondria is common, that selection plays a role in
regulating within-individual allele frequencies, or that
both factors contribute to levels of heteroplasmy in
natural populations. The case for selection is especially
intriguing given that S. vulgaris is gynodioecious, a condition expected to generate strong frequency-dependent
selection on the mitochondrial genome (Ingvarsson
and Taylor 2002; Wade and McCauley 2005).
Mitochondrial Heteroplasmy
TABLE 1
Oligonucleotide sequences
Oligo
atpA forward
atpA reverse
cox1 forward
cox1 reverse
Q forward
Q reverse
Probe X
Probe A
Probe D
59-GTC TGG TCC GAT CGT TTA GC-39
59-TAA AGA GGG CGA TCT TGT CA-39
59-TGG GCA CAT GCT TCT CAG TA-39
59-CTC TTC AAC AGC CCA AGG AC-39
59-ACG TCG CCT GGG AAA GC-39
59-TCC GCG ATA ATG GAA TGC A-39
59-6-FAM-CGG CCT GGT GGT CGG CGT
AAC-BHQ1-39
59-6-FAM-TGA CAT TTG TCG ATA TGC
CAC CG-BHQ1-39
59-HEX-AGA CAT TTG CCG GTA TGC
CAC C-BHQ1-39
Primers and probes for PCR and quantitative PCR of mitochondrial atpA and cox1 that are specific to Silene vulgaris are
shown. Sequences underlined denote cut sites for restriction
enzymes TaqaI, which cuts allele A, and MspI, which cuts allele
D. Bases shown in italics are allele specific. Probe sequences
also include fluorophores with which they are labeled (i.e.,
6-FAM and HEX).
MATERIALS AND METHODS
Genetic material: In the summer of 2004, collections of
leaves and ripe fruit were taken from a number of individuals
from each of three S. vulgaris populations in Virginia. Precise
locations are available upon request. DNA was extracted from
leaves of maternal plants and leaf material from greenhousereared seedlings, using a variation of the CTAB method
(Doyle and Doyle 1997).
PCR/RFLPs: Previous work on S. vulgaris described PCR/
RFLPs in atpA and cox1, using universal primers (McCauley
et al. 2005). All individuals in this study were genotyped for
these PCR/RFLPs, using new species-specific primers that
we have developed to enhance amplification (Table 1). PCR
amplifications were conducted in 50-ml reactions that contained 5 ng of whole genomic DNA, 2 units of Taq polymerase, 125 nm of each primer, 72 mm tricine, 120 mm KCl,
and 4.8 mm MgCl2. Mitochondrial fragments were amplified
using a ‘‘touchdown’’ PCR protocol designed to reduce nonspecific primer associations and subsequent arbitrary fragment amplification (Don et al. 1991). An initial denaturing
cycle of 3 min at 95° was followed by 10 touchdown cycles (the
annealing temperature drops 1° each cycle) of 30 sec at 94°,
30 sec at the annealing temperature of 55°, and 45 or 90 sec,
for atpA and cox1 respectively, at 72° for elongation. The
touchdown cycles were followed by an additional 29 cycles of
30 sec at 94°, 30 sec at the final annealing temperature, and
45 or 90 sec at 72°, which in turn were followed by a 20-min
final elongation period at 72°. Duration of elongation was
determined by the length of the fragment being amplified.
For digestions, 5 ml of PCR product were digested with restriction endonucleases and were visualized on 4% metaphor
gels. Specifically, atpA amplicons were digested with AluI and
MspI in two separate reactions, and cox1 amplicons underwent
a double digest with DdeI and MspI. Representative sequences
of each allele are available in GenBank (accession nos.
DQ422872–DQ422877).
Quantitative PCR: Copy numbers of two distinct atpA lineages within individuals were estimated using the TaqMan
method of Q-PCR (Holland et al. 1991; Lie and Petropoulos
831
1998). These two lineages are very distinct, differing at 16 of
the 665 bp registered with GenBank. Briefly, TaqMan Q-PCR
involves three oligos, a forward primer, a reverse primer, and
a probe. The primers are unmodified and typical of PCR
primers. The probe is designed to target a region within the
sequence to be amplified. It is modified on the 59 end with a
fluorophore and on the 39 end with a quencher. When the
fluorophore and quencher are near to one another, the majority of the fluorescence from the fluorophore is captured.
However, once the fluorophore is separated from the probe by
the exonucleic activity of Taq polymerase, its fluorescence can
escape and be quantified. The intensity of fluorescence is
standardized relative to a passive reference dye. This standardized value is referred to as Rn1: Rn1 -values from the first 15
cycles, prior to any real accumulation of unquenched fluorophore are used to calculate a mean value termed Rn . The
data collected for each cycle of a Q-PCR reaction are DRn
(Rn1 Rn ). The value used to quantify the initial copy number
in a sample is the critical cycle number (CT). The CT is the
estimated number of cycles required for a significant increase
in DRn.
TaqMan probes specific to different S. vulgaris atpA sequences were designed to work with the same primers and were
labeled with different fluorophores such that they could be
used together in a single Q-PCR reaction: the ‘‘A probe’’ for
both the A and B alleles and the ‘‘D probe’’ for alleles D and E.
Of the six atpA alleles previously identified (McCauley et al.
2005), only alleles A, B, and D were found among individuals in
this study. A single-nucleotide difference within the sequence
targeted by a TaqMan probe is generally considered sufficient
for genotyping purposes (Livak 1999). In this study, allelespecific probes were distinguished by three nucleotide substitutions, including one on the 59 end to ensure that probes
function only with their intended alleles (Table 1).
Because several factors influence the rate at which Q-PCR
reactions progress, and because of possible variation in the
sensitivity of hardware to different fluorophores, we designed
a third allele-neutral TaqMan assay, the allele X assay, to
standardize results collected using the A/D assay, Q-PCR using
the A and D probes. The ‘‘X probe’’ is designed to work with the
same primers from the A/D assay; however, the X probe targets
sequence that is invariant across all alleles in this study, and
hence it should function equally well with any allele. The allele
X assay allowed for the standardization of critical cycle (CT)
values obtained using the A and D probes. Both the X assay and
the A/D assay were conducted on 16 individuals for which
allelic identity had previously been established using the PCR/
RFLPs. Eight of these carried the A allele and 8 carried the D
allele. At least five different concentrations of DNA template
were used for both assays. CT was calculated for each individual at each concentration and standard curves were derived.
These data were then utilized to generate Equations 1 and 2
that convert CT-values from alleles A (CTA) and D (CTD) into
comparable copy number estimates:
Log A ¼ ðCT A=2:67Þ 1 8:74
ð1Þ
Log D ¼ ðCT D=2:74Þ 1 8:82:
ð2Þ
All Q-PCR assays were designed using the software Primer
3 (Rozen and Skaletsky 2000) and Applied Biosystem’s
(Foster City, CA) software Primer Express v. 2.0. All primer
and probe sequences are listed in Table 1. All Q-PCR assays
were conducted on an ABI Prism 7000 (Applied Biosystems).
All reaction conditions were optimized according to the protocol for TaqMan Universal PCR Master Mix (Applied Biosystems) with only minor adjustments. Total reaction volume
was 25 ml including 12.5 ml of TaqMan Universal PCR Master
832
M. E. Welch, M. Z. Darnell and D. E. McCauley
Mix (Applied Biosystems). Forward and reverse primer concentrations were 900 nm each, and probe concentrations were
200 nm in all reactions. DNA template concentrations range
from 0.1 ng/ml to 0.8 ng/ml, depending on assay and replicate. CT, the number of cycles required for a significant increase in fluorescence, was estimated with a threshold value of
0.2 using ABI Prism 7000 SDS software (Applied Biosystems).
Primary data collection: Three different Q-PCR experiments were conducted. The first involved running the A/D
assay on all individuals in the study at three different concentrations. The other two experiments were knockbacks that
involve running the A/D assay on three different concentrations of genomic DNA that had each been digested with one
of two specific restriction enzymes. MspI has a single cut site
in the Q-PCR amplicon sequence of allele D, and hence it
prevents its amplification, but not the amplification of the
target sequence in allele A. TaqaI has the opposite effect of
preventing the amplification of the Q-PCR amplicon sequence
of allele A (see Table 1 for specifics). The knockback method
was originally described as a means of revealing cryptic heteroplasmy, using traditional PCR methods, such as might exist
when very unequal copy number limits the PCR amplification
of a minority allele (McCauley et al. 2005). In any PCR, the
exponential rate of the reaction slows and terminates with
increasing product. The knockbacks allow for the necessary
number of functional Q-PCR cycles required for quantifying
minority alleles.
CT-values, estimates of the number of cycles required prior
to a significant increase in PCR product to be detected, were
collected and converted to relative copy numbers, which were
then used to calculate the frequency of allele A within all
individuals. Since a preliminary analysis showed no consistent
difference in allele frequency among the three DNA concentrations that were used in the assays, these were treated as
replicates and averaged. Allele scores were calculated for each
individual using the copy number calculated for A when the
D allele was knocked back and the copy number for D when the
A allele was knocked back.
Analysis of population structure: Under strict maternal inheritance most or all mitochondrial diversity should be partitioned among maternal lines, with little diversity expected
within sibships or individuals. The magnitude of the withinand among-family components of diversity was estimated first
from the PCR/RFLP data. The general principles for calculating genetic diversity indexes are those of Nei (1987) with a
few exceptions that allowed for estimates of genetic variance
attributable to family and individual. First,P
genetic diversity
within families was calculated as Dj ¼ ð1 pij2 Þ, where pij
represents the frequency of allele i within family j. DF, genetic
diversity attributable to family, was then obtained by averaging
Dj over all families. Second, DF was used to calculate the
proportion of genetic variance attributable to differences
among families (Fft ¼ [(DT DF)/DT], where DT is the genetic
diversity found in the entire population), a quantity similar to
the more well-known Fst that quantifies genetic structure
among potentially interbreeding populations. For similar
methodologies of estimating diversity indexes at multiple
levels that take heteroplasmy into account, see Birky et al.
(1989) and Rand and Harrison (1989).
Quantification of mitochondrial heteroplasmy using Q-PCR
allows for the analysis of genetic diversity within and among
individuals in addition to that within and among families
and populations.
P 2 Variation within individuals was calculated
Þ, where pijk represents the frequency p of
as DI ¼ ð1 pijk
allele i in individual k in family j. DI is then averaged for all
individuals. Recall that the Q-PCR method was applied only
to atpA and that the probes could distinguish only two classes
of alleles, the A group and the D group. Note also that the
individual allele scores represent the relative frequency of
the A group (pA) such that qD ¼ (1 pA). Fft was calculated
from the Q-PCR data. Fif (diversity within individuals, relative
to families) was calculated as (DF DI)/DF and Fit (diversity
within individuals relative to the population) was calculated
as (DT DI)/DT.
RESULTS
PCR/RFLP study: The results of the PCR/RFLP
survey are presented in Table 2, with a representative
gel illustrated in Figure 1. A primary allele was readily
scored for all individuals at the atpA locus and all but
four individuals at the cox1 locus. All 4 individuals, 1
mother and 3 offspring from different families, failed to
amplify during PCR using our primers after several attempts, and all are from population 1. However, other
cox1 primers for invariant regions of the gene amplify
product for these individuals, suggesting that variation
in the original primer site prevents amplification in
these individuals and preventing us from scoring two
diagnostic restriction sites for this cox1 allele or class
of alleles. We refer to this cox1 allele as a ‘‘null’’ allele
throughout the remainder of the text. A total of five
atpA/cox1 haplotypes, six if a null allele is accepted,
were distinguished among the 18 mothers and 106 offspring surveyed. Overall, 91 of the 106 offspring studied
(85.8%) resembled their respective mothers. All 15 of
the individuals that did not resemble their respective
mothers were found in population 1, the only population
that was polymorphic for either atpA or cox1. Given that
populations 2 and 3 expressed no PCR/RFLP genetic
variation using these genetic markers, our techniques
should not be able to detect nonmaternal inheritance.
When only population 1 is considered, 61.5% of the offspring resembled their mother if we score the null allele.
Of the 15 individuals that did not match their mothers, 13
differed at cox1 alone, and 2 individuals differed from
their respective mothers at both loci.
Quantification of atpA heteroplasmy: By the Q-PCR
method of atpA genotyping all 18 mothers yielded results in complete agreement with qualitative results of
the PCR/RFLP study when the template DNA was not
subject to the knockback or restriction enzyme treatment and recognizing that the Q-PCR method cannot
distinguish between atpA alleles A and B. The offspring
atpA allele scores from the nonknockback Q-PCR assay
were in agreement with the PCR/RFLP method with six
exceptions. Five individuals, one each from families 1-1,
1-2, and 1-5, and 2 offspring from family 1-6, were found
to be heteroplasmic (allele scores of 0.02, 0.02, 0.01,
0.41, and 0.73, respectively). Thus, by using the Q-PCR
method, 6 of 106 offspring individuals (5.7%) were
shown to have an atpA allele that differed from the
mother, either because it was scored as entirely distinct
from that of the mother or because it showed evidence
of heteroplasmy when the mother did not. All six cases
were found among the 39 offspring scored from
Mitochondrial Heteroplasmy
833
TABLE 2
Haplotypes and allele scores
Maternal haplotype
pi
No. offspring haplotype(s)
1-1
1-2
1-3
1-4
1-5
1-6
1-7
D2
D*
D2
D2
D2
D2
A3
0.03
0.01
0.00
0.29
0.02
0.00
1.00
5 D2, 1 D1
4 D2, 2 D3
6 D2
3 D2, 1 D3, 1 D1
2 D1, 1 D*
1 D2, 1 B1, 1 D*
9 A3, 1 D*
2-1
2-2
2-3
2-4
2-5
A3
A3
A3
A3
A3
1.00
1.00
1.00
1.00
1.00
6
7
7
8
5
A3
A3
A3
A3
A3
0.97–1.00, 0.99 (0.01)
1.00
1.00
1.00
1.00
3-1
3-2
3-3
3-4
3-5
3-6
B1
B1
B1
B1
B1
B1
1.00
1.00
1.00
1.00
1.00
1.00
4
6
6
8
6
4
B1
B1
B1
B1
B1
B1
0.94–1.00, 0.98 (0.03)
0.94–1.00, 0.99 (0.03)
0.85–1.00, 0.97 (0.07)
1.00
1.00
1.00
Population mother
Range, p (SD)
0.05–0.44,
0.01–0.84,
0.00–0.06,
0.00–0.98,
0.03–0.13,
0.22–1.00,
0.01–1.00,
0.23
0.17
0.02
0.30
0.06
0.65
0.89
(0.17)
(0.31)
(0.02)
(0.41)
(0.06)
(0.40)
(0.33)
PCR/RFLP haplotypes and quantitative PCR allele scores for 18 families of Silene vulgaris collected from three
populations (designated 1–3) in Virginia. The atpA allele is designated by letter (A, B, or D) and the cox1 allele is
designated by number (1, 2, or 3). Individuals for which no cox1 allele could be ascribed are denoted with an *.
Offspring not resembling their respective mothers are underlined for emphasis. Q-PCR allele scores for mothers (pi) and the ranges, averages, and standard deviations of offspring allele scores are also presented.
population 1 (i.e., nonstrict maternal inheritance was
found in 15.4% of offspring from population 1). Failing
to find heteroplasmy in populations 2 and 3 using this
method may simply reflect lack of genetic variation in
these populations. Additionally, one of the apparently
heteroplasmic individuals from family 1-6 and one
individual from family 1-4 were scored as allele D by
the PCR/RFLP method, but appear to be predominantly allele A when genotyped using the Q-PCR
method. This dependency of allele on primers used
during PCR was shown to be repeatable.
Application of the knockback technique yielded
rather different results, as illustrated in Figure 2. The
results obtained by calculating an allele score from reciprocal Q-PCR knockback experiments are summarized in Figure 3. Briefly, mothers 1-1, 1-2, 1-4, and 1-5
were classified as heteroplasmic by this method. All
other mothers were scored as homoplasmic. Twenty-six
offspring were scored as heteroplasmic, including the 5
offspring identified as heteroplasmic by the nonknockback method. Twenty-one of the heteroplasmic offspring
were from population 1, 1 was from population 2, and
4 were from population 3.
Within-population structure: Since no variation was
observed within populations 2 and 3 in the PCR/RFLP
study, and little variation was observed in these populations in the Q-PCR study, only population 1 was considered in the following analyses of population structure.
Again, lack of variation in populations 2 and 3 does not
necessarily indicate a lower frequency of heteroplasmy
in these populations, but rather could reflect an issue
of detectability with the markers at hand. From the
PCR/RFLP data presented in Table 2, DT is estimated to
be 0.69, DF ¼ 0.37, and Fft ¼ 0.46, suggesting that on
average 54% (or 1 Fft) of the PCR/RFLP diversity
found in population 1 is contained within families.
See materials and methods for a full description of
summary statistics.
Using the Q-PCR data we get a notably lower index of
total diversity, DT ¼ 0.46, perhaps because the Q-PCR
method distinguished only between two haplotypes instead of six. Diversity within families was comparable to
that of the PCR/RFLP data, DF ¼ 0.27, and diversity
within individuals (DI) was calculated to be 0.12. These
values obtained from the Q-PCR study yield an Fft of
0.41, an Fif of 0.55, and Fit of 0.74. Thus, on average, the
proportion of mitochondrial genetic diversity found
within families that resides within the individuals that
compose those families is 0.45 (or 1 Fif), and, on average, the proportion of variation found in the total
population of offspring that can be found within the individuals that compose that population is 0.26 (or 1 Fit).
DISCUSSION
Heteroplasmy in plants, especially mitochondrial
heteroplasmy, is poorly studied (Korpelainen 2004;
Barr et al. 2005). Extant cases generally fall into two
834
M. E. Welch, M. Z. Darnell and D. E. McCauley
Figure 1.—Agarose gel images of atpA PCR/
RFLPs. Top frames depict atpA amplicons digested with MspI, and the bottom frames are digests with AluI. The three alleles, A, B, and D,
found in this study are represented on the left
(A). Six individuals from family 1-7 are shown
on the right (B). Note that the third individual
from the left carries allele D while its siblings
carry allele A. The mother of family 1-7 also carries allele A.
categories: evidence for recombination has been used
to suggest historic occurrences of heteroplasmy (e.g.,
Städler and Delph 2002; Lin et al. 2003), and experimental hybrids between divergent lineages and
species have provided direct evidence of paternal leakage and heteroplasmy (e.g., Hattori et al. 2002). Mitochondrial heteroplasmy has apparently never been
documented directly in natural plant populations
(Barr et al. 2005) nor has the level of intraindividual
variation been quantified in artificial or natural populations. The only attempt to quantify intraindividual
cytoplasmic genetic variation in plants focused on the
chloroplast genome and did not address issues of
transmission (Frey et al. 2005).
The results presented here confirm findings of an
earlier greenhouse crossing study of mitochondrial heteroplasmy in S. vulgaris (McCauley et al. 2005), extend
them to natural populations, allow heteroplasmy to be
viewed as a quantitative character, and consider the
transmission of heteroplasmy from mother to offspring.
The PCR/RFLP data indicate at least 15 incidences
of mismatch between maternal and offspring mitochondrial alleles, an observation made earlier in the
experimental crosses of McCauley et al. (2005). These
mismatches were all from population 1 and were distributed among six of the seven families sampled. It
is worth noting that the mismatches were observed
using both atpA and cox1 markers, suggesting that this
is a genomewide phenomenon rather than a locusspecific one.
Similarly, in the Q-PCR study most observations of
heteroplasmy were in population 1. In that population
26% of the total mitochondrial diversity occurred
within individuals, whereas variation among families accounted only for 41%, a result not at all consistent with
the very low level of within-individual and within-family
diversity expected to be enforced by strict maternal
inheritance. It is clear that heteroplasmy is a common
feature of at least one natural population of S. vulgaris,
but the mechanism by which it is generated is unknown.
Evidence of heteroplasmy, historical or otherwise, is
most frequently attributed to some degree of biparental inheritance (Mogensen 1996; Korpelainen 2004;
Barr et al. 2005), and this is the most parsimonious
explanation for our findings as well. Several possible
explanations not requiring paternal leakage seem unlikely. The possibility that mutations can accumulate
within individuals faster than they are eliminated or
fixed by maternal inheritance can be dismissed because
16 nucleotide substitutions distinguish alleles A and D,
yet these alleles co-occur within the same individuals.
Selective maintenance of heteroplasmy within strictly
maternal lineages also seems an unlikely explanation
for our data. There are at least nine incidences of mothers yielding offspring with an alternative haplotype in
population 1. Two of these incidences involve mothers
Mitochondrial Heteroplasmy
Figure 2.—Graphical representation of quantitative PCR
(Q-PCR) data. Presented are Q-PCR results from three treatments of the mitochondrial atpA gene for a single offspring
from family 1-1. The y-axis, ln DRn, is a measure of relative
fluorescence, and the x-axis is number of thermal cycles completed. Copy number is estimated from CT, the cycle number
at which a significant increase in fluorescence is detected, approximately the cycle at which curves intersect the shaded
line. Note that copy number for allele A in this individual cannot be estimated when template DNA is not treated with restriction enzymes (Uncut) and that CT for allele D changes
very little when template DNA is undigested (Uncut) and
when template is digested with TaqaI (Cut A) prior to amplification. However, when whole-genomic DNA is treated with
MspI (Cut D), CT for allele A can readily be estimated. Presumably, this occurs because knocking allele D back with MspI
allows for amplification of allele A to continue at an exponential rate for a sufficient number of cycles to be detected. Also
noteworthy is that measurable quantities of allele D persist after treatment with MspI.
that we scored as homoplasmic, giving rise to offspring
that are predominantly of an alternative haplotype. Only
very strong selection favoring the maintenance of multiple distinct mitochondrial lineages could account for
these results given strict maternal inheritance, and this is
in conflict with the largely invariant populations 2 and 3.
Two alternatives involving paralogous loci might also
be invoked to explain some or all of our findings. First,
plant mitochondrial genes are commonly transferred to
the nucleus (Palmer et al. 2000; Richly and Leister
2004). However, in a recent survey of 280 plant genera,
no incidents of loss of mitochondrial atpA or cox1 were
uncovered, suggesting that these genes are not commonly
transferred to the nucleus (Adams et al. 2002). Further,
the experimental crosses conducted by McCauley et al.
(2005) did not find a pattern of inheritance consistent
with nuclear transmission of either of these mitochondrial genes.
Second, the plant mitochondrial genome is complex
and often composed of multiple molecules, and some
secondary molecules, or sublimons, can carry alternate
copies of mitochondrial genes (Backert et al. 1997).
Strong evidence supports the notion that sublimons can
replicate independently of the rest of the mitochon-
835
Figure 3.—atpA allele scores calculated using the Q-PCR
knockback method. Allele scores for 18 mothers (dashes)
and their respective offspring (circles) collected from three
natural populations of Silene vulgaris are shown. Solid circles
represent cases in which two or more individuals from the
same sibship display the same allele score.
drial genome and greatly increase or decrease in copy
number in a single generation, resulting in substoichiometric shifts that could cause an apparent dissimilarity
between mother and offspring ( Janska et al. 1998). This
is not the most parsimonious explanation for our results. Shifts in PCR/RFLP alleles occur at both atpA
and cox1, suggesting that shifts are genomewide and
not gene specific. However, this finding could also be explained by the co-occurrence of both genes on a single
sublimon. Further, some of our shifts in relative frequency are dramatic, minimally five to six orders of magnitude, much greater than the three orders of magnitude
that have previously been documented (ArrietaMontiel et al. 2001). However, given the strong associations between substoichiometric shifting and CMS in
other species ( Janska et al. 1998), and the potential for
this phenomenon to be regulated by nuclear genes
(Abdelnoor et al. 2003), it seems unwise to discount
this explanation entirely.
The results presented here have implications for
other aspects of the population biology of S. vulgaris.
The study of mitochondrial variation in this species
was originally motivated by the fact that it is gynodioecious and displays cytonuclear sex determination
(Charlesworth and Laporte 1998; Taylor et al.
2001), as do many gynodioecious systems (Frank 1989;
Dudle et al. 2001). Much of the theory addressing the
evolution and maintenance of cytonuclear gynodioecy
focuses on the evolutionary conflict between maternally
inherited mitochondrial CMS genes and biparentally
inherited nuclear genes. This intergenomic conflict
could be mitigated by occasional paternal leakage.
Wade and McCauley (2005) have shown that paternal
leakage can have considerable influence on the evolutionary dynamics of cytonuclear gynodioecious systems
836
M. E. Welch, M. Z. Darnell and D. E. McCauley
and can result in a type of frequency-dependent fitness
that contributes to the maintenance of cytoplasmic polymorphism. Crossing studies have shown that S. vulgaris
populations can carry several different CMS elements
(Charlesworth and Laporte 1998; Taylor et al.
2001), an observation that, combined with the results
presented here, suggests the possibility that CMS heteroplasmy could be a factor in the maintenance of
gynodioecy in this system.
The occurrence of heteroplasmy also has implications for the evolution of the mitochondrial genome. In
plants the mitochondrial genome is thought to be structurally quite fluid, owing to a propensity for both intraand intermolecular recombination (Mackenzie and
McIntosh 1999; Palmer et al. 2000). It would seem
that generation of novel recombinants would be facilitated by heteroplasmy, since it would allow for the
pairing of dissimilar DNA molecules during mitochondrial fusion events (Städler and Delph 2002). Indeed,
McCauley et al. (2005) presented evidence for both
inter- and intralocus mtDNA recombination in S. vulgaris, a finding consistent with our observations of frequent heteroplasmy.
The generality of these results to other angiosperm
species is unclear, although the results of the study of
S. acalis by Städler and Delph (2002) suggest that heteroplasmy might be found more generally in gynodioecious members of the genus Silene. The growing
availability of PCR-based molecular markers and Q-PCR
methods for assaying intraindividual genetic diversity
should make investigations of heteroplasmy in a broad
diversity of plant taxa increasingly feasible.
We thank Jonathan Ertelt who provided plant care in the Vanderbilt
University greenhouses. We are extremely appreciative of Douglas
McMahon of the Biological Sciences Department at Vanderbilt University. He allowed us the use of his ABI Prism 7000 for quantitative
PCR, and he and his research group were extremely helpful when
we were designing our Q-PCR assays. This work was funded by a
Vanderbilt University Discovery grant to D.E.M.
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Communicating editor: D. M. Rand